The hepatitis C virus (HCV) is the leading cause of liver cancer in the Western Hemisphere. Over 170 million people are infected with this virus and consequently at increased risk of liver damage and cancer. Understanding how this virus induces liver disease and the development of a much-needed vaccine requires an immunocompetent HCV animal model. The dogma in the HCV field was that this virus only infects humans and chimpanzees. As a recent NIH moratorium on chimpanzee research has effectively prohibited use of these animals, an alternative immunocompetent HCV animal model is desperately needed. To explore if small primates can support HCV infection, we generated hepatocytes from pigtail macaque-(Macaca nemestrina, Mn) induced pluripotent stem cell-derived hepatic cells. Surprisingly, these cells supported detectable levels of infection with cell culture derive genotype 2a HCV. We found that inefficient function of the macaque version of the HCV receptor, CD81, limited HCV infection of these cells. A mutant genotype 2a virus that can use CD81 proteins from a wide range of species overcame this block and efficiently infected the macaque hepatocytes. We have recently made the startling discovery that both wild type and mutant versions of this virus could infect pigtail macaques in vivo, thus demonstrating for the first time that HCV can replicate in animals other than humans and chimpanzees. In this grant we propose experiments to further characterize macaque HCV infections. This will include determining if rhesus macaques, which are more readily available and a larger set of species-specific reagents than pigtail macaques are obtainable, can also be infected with genotype 2a HCV. We will also examine the viral determinants for HCV infection of macaques. We will explore mechanisms by which virus can adapt to more efficiently replicate in this host and determine if we are able to expand the range of viral isolates that can infect NHPs. Ultimately, our studies will provide the first immunocompetent tractable animal models that will establish new avenues to explore HCV replication and pathogenesis in vivo with the eventual goal of developing efficacious vaccine against this virus.